Cells were lysed using RIPA buffer (Solarbio Science & Technology, Beijing, China) supplemented with protease and phosphatase inhibitors (Beyotime, Shanghai, China) for protein extraction. Total proteins were separated on SDS-PAGE gel and transferred to PVDF membranes. Membranes were blotted with primary antibodies for 2 hours at room temperature, membranes were then washed with TBST and incubated in secondary anti-rabbit-HRP (Beyotime) or anti-mouse-HRP antibodies (MDL biotech) for 45 min at room temperature. All protein bands were detected using the ECL reagent (7Sea Biotech) on the Tanon 4200 automatic chemiluminescence image analysis system (Tanon, Shanghai, China).
Ecl reagent
Manufactured by 7Sea Biotech
Sourced in China
ECL reagent is a chemiluminescent detection system used in Western blotting and other immunoassays. It generates a luminescent signal when exposed to peroxidase-labeled secondary antibodies, allowing for the visualization and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (3 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
β actin, by Proteintech (2 mentions)
Enhanced bca protein assay kit, by Beyotime (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Solarbio (1 mentions)
Protease and phosphatase inhibitor, by Beyotime (1 mentions)
Chemiluminescence detection system, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Horseradish peroxidase, by Proteintech (1 mentions)
Hrp conjugated antibodies, by Proteintech (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Hrp goat anti rabbit igg, by Proteintech (1 mentions)
Bovine serum albumin (bsa), by Biosharp (1 mentions)
Gel pro analyzer software, by Media Cybernetics (1 mentions)
Hmgb1, by Proteintech (1 mentions)
Goat anti mouse igg hrp, by Proteintech (1 mentions)
Hmgb1, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
7 protocols using ecl reagent
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of Protein Samples
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Liver tissues (30-50 mg) and cultured cells were prepared in lysis buffer containing 1% of PMSF and 1% phosphatase inhibitor, followed by centrifugation (12,000 × g, 10 min, 4 °C), and the protein concentration was determined by the BCA Protein Assay Kit (P0012, Beyotime, Shanghai, China). Thirty micrograms of protein per lane were separated by 10% SDS-PAGE gels and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes are further blocked with 5% skimmed milk for 2 h at room temperature and incubated overnight with primary antibodies at 4 °C. Then, the membranes were incubated with appropriate horseradish peroxidase (1:5000, Proteintech, China) conjugated secondary antibodies for 50 min at room temperature. The protein blots were detected by enhanced chemiluminescence (ECL) reagent (7 Sea Biotech, China) with a chemiluminescence detection system (Bio-Rad, Chemi Doc, CA, USA). The antibodies used are listed in Supplementary Table 3 .
3
Colon Tissue Protein Quantification and Analysis
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Total proteins extracted from the colon tissues were quantified using a BCA protein assay kit (Beyotime Biotech Co., Ltd., China), separated by SDS-PAGE, and transferred to PVDF membranes (ThermoFisher Scientific). The membranes were incubated with the following primary antibodies: inducible nitric oxide synthase (iNOS), AQP-3 (1:1000, ABclonal, China), C-Kit, ANO1, AQP-4, and AQP-8 (1:1000, Affinity Biosciences) at 4°C overnight. After washing using TBST, membranes were reacted with HRP-conjugated antibodies (1:10000, ProteinTech, USA) at 37°C for 40 min. The bands were monitored with the ECL-Reagent (7 Sea Biotech, China) and visualized with a gel imaging analysis system (LIUYI, China).
4
Protein Quantification and Western Blot Analysis
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Total protein was extracted from the cells by RIPA lysis buffer (Beyotime) containing 1% PMSF (Beyotime) on ice. The concentration of total protein was determined by BCA Protein Quantification Kit (P0009, Beyotime). Protein samples were loaded on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF), the membrane was then blocked with 5% (M/V) dissolved skimmed milk powder for 1 h. Subsequently, the membrane was incubated with anti-ERK (1:1000), anti-p-ERK (1:1000), anti-JNK (1:1000), anti-p-JNK (1:1000), anti-p-p38 (1:1000), anti-p38 (1:1000), anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-cleaved caspase 3/caspase 3 (1:1000) and anti-β-actin (1:2000) antibodies at 4 °C overnight. The incubation of HRP-conjugated secondary antibody (1:10000) was next performed for 45 min at 37 °C after the washing with TBST. The bands were finally visualized with ECL reagent (E003, 7Sea biotech). The optical density of each band was analyzed with Gel-Pro-Analyzer 4. Data were normalized to β-actin.
5
Quantitative Western Blot Analysis
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Total protein extraction was performed using RIPA lysis buffer (Beyotime), with 1% phenylmethanesulfonyl fluoride (PMSF, Beyotime). An enhanced BCA Protein Assay Kit (Beyotime) was used to prepare a standard curve to measure protein concentrations. SDS-PAGE was performed after loading 15 μl protein at 15-30 μg. Once samples were separated, they were transferred to a PVDF membrane (Invitrogen, Carlsbad, CA, USA). The membrane was blocked in 5% albumin bovine (BSA, Biosharp), followed by incubation with diluted primary antibodies at 4°C overnight: HMGB1 (1:1000, Cell Signaling Technology, Danvers, MA, USA), β-actin (1:2000, Proteintech, Wuhan, China). After washing steps, secondary antibodies were added and incubated at 37°C for 40 min: goat anti-mouse IgG-HRP (1:10000, Proteintech) for β-actin, and goat anti-rabbit IgG-HRP (1:10000, Proteintech) for HMGB1. Enhanced chemiluminescence (ECL) reagent (7-Sea Biotech, Shanghai, China) was added to membranes to delineate protein signals, after which membranes were scanned and analyzed using Image acquisition and analysis system (WD-9413B, Liuyi, Beijing, China) and Gel-Pro-Analyzer software (Media Cybernetics, USA).
6
Naringin Modulates NF-κB Activation
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Western blot was performed to analyze the effects of naringin on NF-κB activation. The protein was harvested with RIPA (Beyotime) and quantified with Enhanced BCA Protein Assay Kit (Beyotime) according to the manufacturer’s instructions. Equal amounts of proteins were separated on a SDS- PAGE gel and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). Thereafter, the membrane was blocked with 5% non-fat dried milk for 1 hr. The membrane was incubated with NF-κB p65 primary antibody (1:400; Boster, Wuhan, China) at 4 °C overnight. After washing with TTBS, the membrane was incubated with HRP-labeled Goat Anti-Rabbit IgG(H+L) secondary antibody (1:5000; Beyotime) at 37 °C for 45 min. The membrane was then treated with ECL reagent (7 sea biotech, Shanghai, China) following the manufacturer’s instruction. The protein expression levels were detected using a gel imaging system (Liuyi, Beijing, China). The expression of NF-κB p65 in the cytoplasm was normalized to β-actin and in the nucleus to histone H3.
7
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Beyotime, Shanghai, China) with 1% PMSF (Beyotime) was utilized for total protein extraction. The proteins were denatured at a high temperature and separated by SDS-PAGE. The primary antibodies used are as follows: SOX11 (1:1000; Affinity, Changzhou, China), p-STAT6 (1:1000; Affinity), STAT6 (1:500; Affinity), and β-actin (1:2000; Proteintech, Wuhan, China). Antibody binding was visualized using secondary antibodies (Proteintech). The blots were analyzed using Gel-Pro-Analyzer software with the ECL reagent (7sea biotech, Shanghai, China).
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